The cholecystokinin (CCK) receptor is a family A peptide-binding G protein-coupled receptor that has multiple important physiologic functions supporting nutritional homeostasis, including the stimulation of pancreatic exocrine secretion and gallbladder emptying, the regulation of gastric emptying and bowel transit, and the modification of appetite. Like many other members of this superfamily, this receptor is a potentially important drug target, based on its role to induce satiety that can be useful in the prevention and/or treatment of obesity. The long-term goal of the current proposal is to enable more rational and directed CCK receptor drug development, built upon our recent observations that distinct small molecule CCK receptor ligands can have an allosteric mode of action and that the lipidic microenvironment of the type 1 CCK receptor can lead to dysregulation of receptor signaling. The proposed projects fall under two major aims.
The first aim i s """"""""receptor-centric"""""""", directed toward the elucidation of the structure of the small molecule-binding pocket within the helical bundle of the receptor and the chemistry of ligands binding there to achieve allosteric enhancement of the action of CCK. There are three sub-aims directed toward the following: (1.1) gaining detailed structural insights into the molecular basis of selectivity for the types 1 and 2 CCK receptors using mutant and chimeric receptors;(1.2) gaining insights into the determinants for biological activity of small molecules acting within this pocket using receptor mutagenesis;and (1.3) characterizing conformational changes in the cytosolic effector face of this receptor correlating with full agonist, partial agonist, and biased agonist activities using novel fluorescence techniques.
The second aim i s """"""""environment-centric"""""""", directed toward the lipid microenvironment of the CCK receptor and its impact on type-selective CCK stimulus-activity coupling. There are three sub-aims directed toward the following: (2.1) determining the structural basis for cholesterol sensitivity of the type 1 CCK receptor using mutant and chimeric receptors;(2.2) developing stable model cell lines having elevated membrane cholesterol and cell impermeant fluorescent probes of membrane cholesterol composition that can be utilized for morphological analysis, and that can be applied to flow analysis and sorting of living cells;and (2.3) using these reagents to more fully characterize the impact of an abnormal membrane lipid environment on type 1 CCK receptor function, and to develop and validate a strategy to normalize CCK stimulus-activity coupling in the setting of excess membrane cholesterol. Together, these activities should reenergize efforts to target the CCK receptor for the prevention and treatment of obesity, providing strategies for the development of novel allosteric enhancers that could be safer and more effective than existing full agonist candidate drugs, thereby exerting a sustained and powerful influence on a major public health problem.
While obesity is a major public health problem affecting an increasingly larger proportion of the American population, effective prevention and long-term management is lacking. This project is directed toward the development of new, safer, and more effective types of drugs acting at a receptor known to regulate appetite, the cholecystokinin receptor. Component aims are directed toward structural and mechanistic analysis of a drug-binding pocket within this receptor and toward better understanding of the impact of the cholesterol composition of the cell membrane in which this receptor resides.
|Desai, A J; Dong, M; Harikumar, K G et al. (2016) Cholecystokinin-induced satiety, a key gut servomechanism that is affected by the membrane microenvironment of this receptor. Int J Obes Suppl 6:S22-S27|
|Miller, Laurence J; Desai, Aditya J (2016) Metabolic Actions of the Type 1 Cholecystokinin Receptor: Its Potential as a Therapeutic Target. Trends Endocrinol Metab 27:609-619|
|Desai, Aditya J; Dong, Maoqing; Miller, Laurence J (2016) Beneficial effects of ?-sitosterol on type 1 cholecystokinin receptor dysfunction induced by elevated membrane cholesterol. Clin Nutr 35:1374-1379|
|Desai, Aditya J; Henke, Brad R; Miller, Laurence J (2015) Elimination of a cholecystokinin receptor agonist 'trigger' in an effort to develop positive allosteric modulators without intrinsic agonist activity. Bioorg Med Chem Lett 25:1849-55|
|Harikumar, Kaleeckal G; Miller, Laurence J (2015) Use of Fluorescence Indicators in Receptor Ligands. Methods Mol Biol 1335:115-30|
|Desai, Aditya J; Lam, Polo C H; Orry, Andrew et al. (2015) Molecular Mechanism of Action of Triazolobenzodiazepinone Agonists of the Type 1 Cholecystokinin Receptor. Possible Cooperativity across the Receptor Homodimeric Complex. J Med Chem 58:9562-77|
|Desai, Aditya J; Dong, Maoqing; Harikumar, Kaleeckal G et al. (2015) Impact of ursodeoxycholic acid on a CCK1R cholesterol-binding site may contribute to its positive effects in digestive function. Am J Physiol Gastrointest Liver Physiol 309:G377-86|
|Dong, Maoqing; Vattelana, Ashton M; Lam, Polo C-H et al. (2015) Development of a highly selective allosteric antagonist radioligand for the type 1 cholecystokinin receptor and elucidation of its molecular basis of binding. Mol Pharmacol 87:130-40|
|Desai, Aditya J; Harikumar, Kaleeckal G; Miller, Laurence J (2014) A type 1 cholecystokinin receptor mutant that mimics the dysfunction observed for wild type receptor in a high cholesterol environment. J Biol Chem 289:18314-26|
|Dong, Maoqing; Miller, Laurence J (2013) Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling. Peptides 46:143-9|
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